Rodent models and accompanying in vitro strategies for studying the etiology and therapy of Parkinson's disease

Abstract

The lack of disease-modifying therapies for Parkinson’s disease (PD) places a severe burden on patients, their caregivers and aging societies. While the incomplete knowledge of underlying causes and pathophysiology remains a major bottleneck towards the development of rational therapies, there have been important advances in our understanding of neurodegenerative processes. Here, animal models have contributed substantially by allowing researchers to decipher PD-relevant pathology and the development of corresponding behavioral signs, which enabled development and refinement of treatment options. Without animal models, hypotheses of mechanisms underlying the development of motor symptoms after dopamine depletion, dyskinesia as side effects, the impact of diverse environmental neurotoxins, or the formation of alpha-synuclein pathology and spreading could not have been conclusively tested. Likewise, novel technologies are enabling the use of patient derived tissues for generating in vitro models that further enhance our understanding of mechanisms and of the effects of specific genetic modifications and their role in neurodegeneration. However, animal models are, as of yet, still required to model a fully functional central nervous system connected to the immune system and peripheral organs, which are impacted by the diversity of PD pathology and symptoms. Here, we provide an overview of key contributions of animal models, their ongoing role in PD research, and how this research can be enhanced by the concomitant use of advanced in vitro systems.

Plain language summary

Animal models and cell models to study Parkinson's disease

There is currently no treatment that can slow or stop the progression of Parkinson's disease (PD), which places a heavy burden on patients, caregivers, and aging societies. One major reason for this is that we still do not fully understand what causes PD. However, recent research has improved our understanding of the underlying disease processes. Animal models have played a crucial role in this progress. They allow scientists to study how PD-related loss of neurons lead to symptoms, and they have helped in developing and improving treatments. Without animal models, we would not be able to test important ideas about how dopamine loss leads to motor symptoms, why certain treatments cause side effects like dyskinesia, how environmental toxins contribute to the disease, or how abnormal proteins like alpha- synuclein spread in the brain. New models now let researchers study cells and tissues taken from patients. These tools make it easier to explore how specific genetic mutations contribute to brain cell damage. Still, animal models are currently the only way to study how Parkinson's affects the whole nervous system, including its connections to the immune system and other organs, all of which can be involved in the disease. This article reviews the important contributions of animal models to Parkinson's research, their ongoing value, and how combining them with advanced in vitro systems can improve our understanding of the disease and lead to better treatments.

Keywords

animal models in vitro organoids Parkinson's disease neurodegeneration

Introduction: The growing burden of Parkinson’s disease

Parkinson's disease (PD) is the second most frequent progressive neurodegenerative disorder.¹ According to the World Health Organization there are about 8.5 million individuals suffering from PD in 2019.² Worryingly, PD is the fastest growing neurological disease in aging societies, and the global prevalence is expected to further double within the next 20 years (up to 2% in people over the age of 60 and 6% in people over 80 years).² PD is defined as a motor syndrome with dopamine neuron loss in the substantia nigra pars compacta, accompanied by aggregation of the protein alpha-synuclein (aSyn) in Lewy bodies.^3,4 Since neurodegeneration and protein accumulation can only be detected postmortem, the clinical diagnosis relies on characteristic motor symptoms of Parkinsonism that include bradykinesia, resting tremor, rigidity, and postural instability.⁴ These symptoms are mainly caused by the progressive dopaminergic neurodegeneration and consequently, lack of the movement controlling neurotransmitter dopamine. Alterations in non-dopaminergic neurotransmitter systems add to the complexity of the disease and are associated with non-motor symptoms.^5–8 Importantly, these symptoms, such as olfactory deficits, rapid-eye-movement sleep behavior disorders, and autonomic dysfunction, precede the overt motor deficits by years or even decades.⁹ While their occurrence may indicate an increased risk to develop PD, they do not lead to reliable risk assessments and are not validated as endpoints for clinical trials.

The available dopamine replacement therapy efficiently reduces the motor symptoms, but does not slow down disease progression, does not treat most non-motor symptoms and is limited in dose and duration due to the development of L-DOPA induced dyskinesia. There is currently no therapy available to prevent, halt or cure this debilitating disorder, which places a very high burden on patients and caregivers. At time of diagnosis patients are still highly engaged and active members of our society, part of the work force and central to the well-being of their families. The economic burden of PD solely for the US has been estimated at around $51.9 billion for 2017, including direct and indirect medical costs, reduced employment, disability income, and reduced loss in volunteer work.¹⁰ In Europe, the total costs for PD alone in 2010 amounted to €13.9 billion.³

These facts underline that significant efforts are necessary in order to develop disease-modifying treatments. However, this requires further research into the disease pathomechanisms, discovery of novel drug targets, and drug development towards clinical trials. To achieve this, researchers need to conduct basic science studies, using a variety of model organisms and systems. While enormous efforts have been focused on replacing in vivo experiments in animals by using ex vivo and in vitro systems, the fact is that there is still no alternative to animal models for establishing drug efficacy and safety prior to clinical trials in human subjects. The urgency to develop treatment provides the ethical backbone for the use of animal models, strictly following the 3R principles, which are now part of good laboratory and scientific practice in research. There have been several reviews providing in-depth overviews of animal and cellular models of PD, including by our groups. Therefore, here we discuss key findings (summarized in Table 1) that were learned from using animal models to highlight their essential role in PD research and how they enabled numerous benefits for patients. Furthermore, we discuss complementary ex vivo and in vitro approaches to highlight their potential as well as their current limitations to reduce, refine or replace in vivo experiments.

Table 1.

Key learnings from commonly used animal models of PD.

Key learnings	Model / toxin	Mechanism	Pathology and symptoms	Further observations
Dopamine is responsible for many PD-associated symptoms	Reserpine (rodent model)	Inhibits vesicular monoamine transporter-2 (VMAT)¹¹	Transient dopamine loss^12–14 Levodopa-reversible akinesia, rigidity¹⁵ Sexually dimorphic motor deficits¹² Cognitive deficits, sleep abnormalities, anxiety, depressive-like behavior, gastrointestinal dysfunction¹⁶	No aSyn pathology¹⁷ No mitochondrial or lysosomal dysfunction¹⁸ No inflammation¹⁸
Dopamine is responsible for many PD-associated symptoms	Haloperidol (rodent, non-human primates)	Inhibition of post-synaptic dopaminergic D2 receptors¹⁹ → Dopamine transmission blockade²⁰	Reduced dopamine, noradrenaline and serotonin²¹ Mitochondrial complex I deficit²² Increased cytokine levels²³ Rigidity, dyskinesia, and catalepsy²⁰	No dopaminergic neurodegeneration²⁴ No aSyn pathology
Pesticides increase risk of PD via mitochondrial damage and oxidative stress	6-Hydroxydopamine (6-OHDA) (C. elegans, D. melanogaster, rodents, non-human primates)	Production of ROS and mitochondrial complex I inhibition^25,26	Dopaminergic neurodegeneration and axonal denervation^27,28 Motor deficits²⁷ Depression-like behavior²⁹ Gastrointestinal dysfunction^30,31	Intracranial injection necessary²⁷ No aSyn pathology²⁴ Pathology limited to injection site²⁷
	1-Methyl-4-phenyl-1,2,36-tetrahydropyridine (MPTP) (rodents, although rats are less sensitive, non-human primates)	Acute ATP deficit, ROS production via mitochondrial complex I inhibition³²	Dopaminergic neurodegeneration³³ Microglia activation and cytokine release³⁴ aSyn and tau pathology³⁵ Tremor, rigidity, bradykinesia, postural instability, freezing³⁶	High variability depending on dosage, species and strains³⁷
	Rotenone (C. elegans, D. melanogaster, rodents)	Mitochondrial complex I inhibition³⁸	Dopaminergic neurodegeneration³⁹ aSyn and tau pathology^39–41 Oxidative stress^38,40 Microglia activation³⁸ Impaired autophagy^38,42 Hypokinesia, rigidity, reduced locomotion and rearing^39,41,43	High inter-individual variability⁴⁴
	Paraquat and Maneb (C. elegans, D. melanogaster, rodents)	Metabolite PQ2 + penetrates dopaminergic neurons via DAT⁴⁵	Dopaminergic neurodegeneration^46,47 Transient aSyn pathology⁴⁸ Microglia activation⁴⁶	Pulmonary fibrosis as side effect⁴⁹
Infections are risk factors for neurodegenerative diseases by driving inflammatory and protein-aggregating processes	Lipopolysaccharide (LPS) (rodents)	Toll-like receptor 4 driven microglia activation⁵⁰	Dopaminergic neurodegeneration⁵¹ Mitochondrial complex I and II dysfunction⁵² aSyn pathology⁵³ Microglia activation and cytokine release⁵⁴ Hypokinesia, rigidity⁴⁶ Motor deficits⁵⁵	Phenotype depends on application route, age of animal, species and strain⁵⁰
PRKN- and PINK1-regulated pathways of mitochondrial function are involved in PD pathogenicity	PRKN knockout (C. elegans, D. melanogaster, rodents)	Mitophagy disruption⁵⁶	Mitochondrial dysfunction⁵⁶ Motor deficitis⁵⁷	No aSyn pathology⁵⁸
	PINK1 knockout (C. elegans, D. melanogaster, rodents, non-human primates)	Mitochondrial quality control pathway⁵⁶	Mitochondrial dysfunction⁵⁶	No aSyn pathology⁵⁸ Age-dependent phenotype⁵⁹
LRRK2 kinase inhibitors are a potential therapeutic target	LRRK2 G2019S overexpression / mutation (C. elegans, D. melanogaster, rodents)	Enzyme involved in autophagy, mitochondrial function, vesicular trafficking⁶⁰	Dopaminergic neurodegeneration^61,62 aSyn and tau pathology^63,64 Autophagic and mitochondrial dysfunction⁶⁰ Hypoactivity^61,65 Impaired memory function⁶¹
Protective effect of DJ-1 against oxidative stress is lost in some PD patients	DJ-1 knockout (D. melanogaster, rodents)	Increased oxidative stress via mitochondrial dysfunction⁶⁶	Dopaminergic neurodegeneration⁶⁷aSyn pathology⁶⁸Motor deficits⁶⁷	Age dependent phenotype⁶⁷
Dysfunction of protein-degradation is involved in PD pathology	UCH-L1 mutation (D. melanogaster, rodents)	Ubiquitin-proteasome pathway⁶⁹	Axonal degeneration^70,71Mitochondrial dysfunction⁷²Dopaminergic neurodegeneration⁷³	No aSyn pathology⁷³
	GBA1 mutation (C. elegans, D. melanogaster, rodents)	Lysosomal glucocerebrosidase enzyme → accumulation of glycosphingolipids⁷⁴	aSyn pathology⁷⁵Microglia activation⁷⁵Reduced striatal dopamine⁷⁵Mitochondrial/autophagic dysfunction⁷⁶Motor deficits⁷⁷	Often combined with aSyn overexpression to increase aSyn accumulation⁷⁸
aSyn aggregation plays an important role in PD pathophysiology	Wildtype aSyn overexpression mouse model	Overexpression of wildtype aSyn⁷⁹	Mitochondrial dysfunction^80,81Neuroinflammation^80,81aSyn pathology⁸²Striatal denervation⁸³Motor deficits^83,84Non-motor symptoms^83,84	DA loss in striatum, but only DA neuron shrinkage in SNc⁸³Age dependent phenotype⁸³
Posttranslational modifications increase aSyn toxicity	C-terminally truncated aSyn mouse model	Increased aggregation properties compared to wildtype aSyn⁸⁵	aSyn pathology⁸⁵Motor deficits⁸⁵	Age dependent phenotype⁸⁵
	E46K human aSyn mouse model	Increased aggregation properties compared to wildtype aSyn⁸⁶	aSyn pathology⁸⁷Astrocytic gliosis⁸⁷Tau inclusions⁸⁷	Age dependent phenotype⁸⁷No dopaminergic neurodegeneration
	A30P human aSyn mouse model	Increased aggregation properties compared to wildtype aSyn⁸⁸	aSyn pathology⁸⁸Mitochondric/autophagic dysfunction⁸⁹Locomotor disabilities⁸⁸	Neuronal cell loss inconsistent²⁴
	A35T human aSyn mouse model	Increased aggregation properties compared to wildtype aSyn⁹⁰	aSyn pathology⁹⁰Motor deficits⁹⁰	Age dependent⁹⁰
aSyn fibrils can propagate in a prion like manner and pathology depends on aSyn fibril strain used	aSyn preformed fibrils (PFFs) (mice, non-human primates)	PFFs phosphorylate endogenous aSyn into pathological state⁹¹	Enhanced aSyn spreading⁹¹Degeneration of striatal fibers⁹²Inflammation⁹³Motor deficits and end-stage paralysis^92,93	Depending on substrate and injection site²⁴
	aSyn viral expression (AAV/lentivirus) (rodents, non-human primates)	Region-targeted aSyn overexpression^94–96	Dopaminergic neurodegeneration⁹⁷Lewy body-like structures⁹⁷Inflammation⁹⁸Mitochondrial dysfunction⁹⁹Autophagy¹⁰⁰Motor deficits¹⁰¹	Depending on virus titer, injection site, strain and species influence outcome¹⁰²
Neuromelanin is a potential mediator of neurotoxicity in PD	Neuromelanin-like production (rodent model)	Protective and toxic effects of neuromelanin¹⁰³	Microglia activation¹⁰⁴Autophagy¹⁰⁵Impaired dopamine homeostasis¹⁰⁵Lewy body-like structures¹⁰⁵Motor deficits¹⁰⁵	Depends on synthetic neuromelanin injection or human tyrosinase production^104,105

Key learnings from animal models regarding the pathomechanisms of PD

Toxins, pesticides and infections contribute to PD risk

Apart from aging, there are several risk factors that contribute to PD and its increasing prevalence.¹⁰⁶ According to the multiple hit theory, PD is the consequence of additive risks acquired during lifetime.^107,108 These insults include genetic risk factors, but also intrauterine and peri-/postnatal infections, as well as exposure to neurotoxic herbicides or pesticides.^86,109–114 While risk can be assessed in epidemiological studies, it is still not possible to accurately track pathomechanisms in patients, in absence of biomarkers, brain imaging markers, and access to brain tissue. Thus, we learned about the impact and mechanisms of risk factors from work with animal models.

A striking example comes from a major breakthrough in PD research, with the discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP), an impurity in desmethylprodine solutions synthesized and injected by drug addicts, induces a persistent parkinsonian syndrome in humans.^113,115 As a lipophilic compound, MPTP crosses the blood brain barrier and is converted by astrocytic MAO-B into MPP+, which is selectively taken up into dopaminergic neurons via the dopamine transporter. Within mitochondria, MPP + inhibits complex I, mimicking key aspects of PD pathology.^116–118

Most of these mechanisms were uncovered in mice and non-human primates, which are sensitive to MPTP toxicity, whereas rats are relatively resistant.¹¹⁹ These observations led to research into mitochondrial deficiency in PD patients, and the role of oxidative stress in the pathogenesis of PD, which both are now accepted as key pathogenic pathways. Follow up work in mice and rats using pesticides such as paraquat and rotenone uncovered that the increased risk of developing PD if exposed to these environmental toxins is due to complex pathomechanisms including mitochondrial inhibition and increased oxidative stress. Of note, the risk for neurodegeneration increases if the pesticide exposure is combined with certain genetic predispositions, such as changes in the expression of the dopamine transporter.^120–123 This led to re-evaluation of the use of pesticides across the world. An important aspect of the specific susceptibility of dopaminergic neurons to oxidative stress is the presence of neuromelanin, a by-product of toxic dopamine metabolism which accumulates with aging. Over-expression of tyrosinase in the rat substantia nigra has been used to induce neuromelanin deposition,¹⁰⁵ confirming its toxicity for dopaminergic neurons.

More recently, there has been renewed focus on the role of infections as risk factors for PD. Similar to low-dose toxin exposure, the risk increase is likely compounded over the lifetime, and very difficult to track in humans. We recently contributed to a review of the current literature, demonstrating significant evidence for viral and bacterial infections to increase PD risk.¹²⁴ Again, part of this evidence, and specifically insights into the underlying mechanisms, were derived from animal models. For instance, it was hypothesized that infections with influenza virus, West-Nile virus, picornavirus, coxsackievirus or SARS-CoV-2 could be associated with or even cause aSyn pathology.^124–126 While SARS-CoV-2 is generally not a neurotropic virus, we and others demonstrated that intranasal infection in hamsters can cause persistent inflammation across the brain, accompanied by tau and aSyn pathology and loss of GABAergic interneurons, with sex-specific pathology patterns.^127,128 These findings could, at least in part, explain how viruses could cause neurological symptoms in the absence of severe neuroinvasion. Repeated exposure to viruses could, similarly to environmental toxins, increase the risk to initiate neurodegenerative processes. If these processes are self-propagating, they would become unmasked as progressive neurodegenerative disease with aging.

Although the precise underlying mechanisms require further research, nearly all studies on infection and PD demonstrate a role of complex immune responses, requiring communication between the peripheral and the brain-resident immune cells across the blood brain barrier. For instance, during gastrointestinal dysbiosis, the bacterial endotoxin lipopolysaccharide (LPS) can enter the blood stream, leading to an acute peripheral immune response followed by microgliosis in the brain, which in turn may impact neuronal function.^129–133 These complex mechanisms highly relevant to the human disease require an intact organism to study, and while there are significant differences in the cellular immune systems makeup, mice are established and well-characterized models to study the immune response. Especially the possibility to genetically modify specific immune cells and receptors in mice enables proof of concept studies.¹³⁴ Furthermore, testing any type of drug interfering with the immune response requires careful efficacy and safety studies in vivo in order to exclude any unexpected deleterious target or off target effects on the brain or other organs.

How aSyn may be detrimental to neurons in PD

Cytoplasmic deposits and dystrophic neurites, so-called Lewy bodies and Lewy neurites, are immunopositive for the protein aSyn and represent the histopathological hallmark of PD.¹³⁵ Lewy body pathology has been suggested to spread in the brain, and also outside the brain, during disease progression, and assessing its distribution is a common method to evaluate the stage of disease in postmortem brain tissue (Braak staging system).^136,137

aSyn is a small 14 kDa and 140 amino acid long protein, and its accumulation characterizes PD as a synucleinopathy.¹³⁸ In its native form, aSyn lacks defined tertiary structure and, therefore, has been described as an intrinsically disordered protein (IDP).^139,140 However, in synucleinopathies the protein forms aggregates rich in β-sheet structures that can propagate from cell to cell in a prion-like manner.^141,142

Several different posttranslational modifications (PTMs) can be found in aSyn in postmortem PD brains (e.g., phosphorylation at Serine 129 (pS129), ubiquitination, C-terminal truncation, or glycation).^143–146 While these observations from patient brains support an important role of aSyn in PD pathophysiology, the underlying mechanisms have been discovered and studied in disease models. In mice, it was shown that increased aSyn expression is sufficient to cause its aggregation in neurons, with concomitant neuronal dysfunction, similar to what is thought to happen in PD.^82,147,148 Furthermore, overexpressing mutated familial forms of aSyn causes PD-like pathology and symptoms in mice, supporting a gain of toxic function, and the proteinopathy hypothesis.^90,149

Studies in mouse models demonstrated that recombinant aSyn fibrils or aSyn aggregates/ Lewy bodies derived from human brains can propagate in a prion like manner from neuron to neuron or even from the gut to the brain, when injected intracerebrally or peripherally.^92,150–152 Furthermore, work in mouse models contributed to the discovery that different aSyn fibril strains induce specific synucleinopathies.¹⁵³ These studies uncovered multiple potential pathogenic pathways involved, such as the induction of lysosomal and mitochondrial dysfunction. In addition, they provided in vivo evidence that oligomeric forms of aSyn may be the most toxic forms to neurons.¹⁵⁴

Lysosomal dysfunction and increased PD risk

As for all other proteinopathies, pathways associated with protein clearance are thought to play a major role in PD. For example, studies of the ubiquitin-proteasome system showed that some of its components are expressed less, and also that its catalytic activity is reduced in PD.¹⁵⁵ Furthermore, autophagy-lysosomal dysfunction is involved in PD pathophysiology, given that mutations in lysosomal genes, an accumulation of autophagic vacuoles, and reduced levels of lysosomal proteins are associated with an increased PD-risk.^156–158 The discovery that Gaucher disease patients or carriers of mutations in the gene encoding glucocerebrosidase (GBA) have a higher risk of developing PD substantiated lysosomal dysfunction as relevant pathway. Follow up work in cell culture proposed a positive feedback loop between aSyn accumulation and lysosomal dysfunction.^159–161 Lysosomal dysfunction is a common observation in toxin-induced and genetic models of PD, and has been extensively studied. Furthermore, cross-breeding of animal models of lysosomal storage disorders, with PD mouse models, demonstrated increased PD pathology, as a proof of concept. These animal models were used to develop and test novel therapeutic strategies, of which some are currently tested in clinical trials.^162–167

Key learnings from animal models regarding current PD therapies

Refinement of symptomatic dopamine replacement therapy

The relationship between motor symptoms and the dopaminergic system was first demonstrated in 1957 via reserpine administration, which induced an akinetic state associated with catecholamine depletion in the central nervous system. This state could be reversed by L-DOPA administration,^15,168 laying the foundation for all subsequent insights into PD pathophysiology. However, reserpine does not kill dopaminergic neurons, limiting its use to study therapeutic interventions.

The neurotoxin 6-hydroxydopamine (6-OHDA) selectively targets catecholaminergic neurons via monoamine transporters, inducing neurodegeneration.^169,170 The discovery of the effects of this neurotoxin in animals was instrumental for the field. As a hydrophilic molecule, 6-OHDA cannot cross the blood brain barrier and must be injected stereotaxically—either intraventricularly, intrathecally, or intracerebrally. Intracerebral injection allows targeted, unilateral or bilateral application.^171,172 Its toxic effects are thought to result from oxidative stress due to its metabolic similarity to dopamine.^173,174 The classic unilateral 6-OHDA injection model produces rotational behavior after dopaminergic stimulation (e.g., apomorphine, amphetamine).^175,176 As the model lacks progressive degeneration as well as aSyn inclusions, it is not suitable for developing and assessing disease-modifying therapies. However, it has been indispensable to refine dopamine replacement therapy, especially as rats and mice develop dyskinesias under L-DOPA treatment, similar to PD patients.^177–179

Challenges in developing disease-modifying therapies

In contrast to symptomatic treatment, disease-modifying therapies are meant to target neurodegenerative processes, in order to halt or prevent progression.^180,181 However, promising therapeutic candidates failed to reach the primary endpoints defined in the trials - improvement of motor function.¹⁸² Strikingly, all of these compounds did, at some stage of development, show efficacy in animal models and, therefore, this lack of predictive validity is a common argument against the use of animal models. Nevertheless, the role of these models is to enable the testing of research hypothesis, and to provide the proof of concept that the drug reaches and engages its target.

In pre-clinical work, animal models that replicate the targeted signaling pathway and pathophysiology are chosen, and if the drug engages its target, these models will provide the respective positive response. As an example, models based on the injection of aSyn pre-formed fibrils (PFFs) as described above, model the formation and prion-like spreading of aSyn pathology across neurons. Drugs that directly interfere with oligomerization or misfolding of aSyn could be tested in these models. However, whether this will be sufficient to reduce the already ongoing multifaceted pathology in dopaminergic neurons of clinically diagnosed PD patients, is questionable. Likely, if the same compound is tested in a model of mitochondrial deficiency, or microglia activation, it will not result in positive outcome.

The important take home message from failed clinical trials is, that neuroprotection in PD will require treating very early in disease progression, long prior to clinical diagnosis, and/or developing strategies of drugs that target several pathogenic pathways, or combining drugs with complementary, additive or even super-additive effects. In any case, the animal models used need to be carefully chosen to replicate the targets to be engaged.⁸⁴ A single model should not be expected to encompass such a multi-system, multi-faceted and multi-mechanistic disease, it should at least replicate nigrostriatal dysfunction and motor symptoms to predict main clinical endpoints, together with aSyn pathology and microgliosis.^82,183,184

In recent years, molecular changes were proposed to be the more valid endpoint compared to behavioral signs, however, this did not lead to success in clinical trials. Instead, it may be most important to understand which molecular pathway alteration in which brain region is linked to a specific behavioral readout. In toxin induced models, this relationship is straight forward, e.g., dopamine loss is linked to motor deficits in the 6-OHDA model described above. In more slowly progressing synucleinopathy models with pathology across peripheral and central neurons, involving multiple pathogenic mechanisms, this direct link between the construct and face validity is more challenging to decipher.^82,183,184

Another often overlooked aspect of the role of animal models are the many drugs that do not make it to the clinics because their effects were deleterious in animal models. If the prediction of clinical benefit for a drug target is built on an incomplete picture of its role in neuronal function, the drug could have unexpected deleterious on-target effects. Consistently, we recently showed that mice over-expressing aSyn replicated the clinical failure of venglustat, thereby providing possible mechanisms for the observed worsening of motor symptoms in patients.¹⁶⁶ The bulk of experimental evidence for venglustat to move to the clinics was derived from advanced in vitro models, and the results were valid in these young cells accumulating lysosomal substrates. However, they did not translate into clinical benefits.

The reasons why the various strategies tested thus far showed no improvement of motor function in PD patients can be manifold and are, for the most part, still unclear. Nevertheless, failed trials also offer tremendous opportunities for learning. For instance, they suggest that the impact of tested therapeutic targets on the degeneration of dopaminergic neurons at the stage of disease when the clinical trial is conducted may have been overestimated. The degenerative processes may have been too far advanced: e.g., mitochondrial dysfunction, lysosomal dysfunction, build-up of aggregated proteins, inflammatory processes. In addition, failed trials suggest that halting one signaling pathway in one of these processes is likely not sufficient to improve dopamine neuron function such that the clinical outcome improves significantly.

Studies involving animal models remain instrumental for moving into clinical trials, even if the FDA is making some exceptions for some conditions. Currently there are a number of Phase 2 and 3 trials with PD therapeutics in the planning stages or ongoing,¹⁸⁵ with a few disease-modifying strategies. The diagnosis of PD has recently shifted the focus on underlying disease biology.¹⁸⁶ If quantifiable biomarkers and brain imaging will become applicable as clinical endpoints, we will be able to further refine in vivo efficacy studies to increase predictive validity and to reduce the numbers of required animals.

Ex vivo and in vitro strategies for assisting and reducing the use of animals

Strategies for reducing or replacing animal experiments are evolving in the field of PD research. While cellular models cannot reproduce the broad complexity of PD pathophysiology, they enable modelling the cellular phase of the disease via specific gene manipulations, and the study of various molecular processes such as aSyn pathology, oxidative stress, autophagy, or apoptosis. Importantly, they are also suitable for high throughput screenings.^186,187 Therefore, they offer a highly valuable toolbox for assisting and guiding animal experiments, thereby increasing our understanding of disease mechanisms and ultimately enabling the identification of suitable targets for therapeutical intervention (Figure 1). By rapidly screening therapeutic targets and candidate molecules instead of using animals for these efforts, in vitro methods ease ethical concerns raised by the use of experiments in mammalian systems, while also contributing to reduce cost and to save time.

Figure 1.

Example of combined uses of in vitro and in vivo models during drug screening. In vitro and ex vivo PD models include immortalized cell lines, primary neurons, 3D-structured organotypic brain slices, iPSCs and organoids. Especially, regarding cellular and molecular mechanisms of disease progression, focusing solely on cells instead of whole organisms enables a deep analysis of cellular pathways. Functional assays to measure (I) cell death in context of neurotoxicity due to protein accumulation or dopamine-derivates, (II) neuronal firing rate to assess functionality of neurons, and (III) tracking of organelles to study protein degradation or mitochondrial dysfunction more precisely. Evolving in vitro methods are suitable for high throughput screening of drugs since access to cell models is easier and ethical concerns are avoided. They should be seen as additional tools to investigate mechanisms of PD and identify suitable targets for therapeutic intervention. Animal models will always be a crucial part in PD research, to determine progression of motor symptoms and long-term effect of treatment options using motor tests. Figure created with BioRender.

In vitro models can be very useful especially when studying cellular mechanisms and cellular pathways related to cytotoxicity or neuronal network degeneration during PD progression.¹⁸⁸ Moreover, novel in vitro assays also allow quantification of neuronal firing rates, neuronal axon and network growth, lysosomal tracking, and mitochondrial functionality. All of these approaches enable in-depth investigations of cellular pathways, easily overlooked when using a whole organism. For example, immortalized cell lines that resemble human dopaminergic neurons and primary neurons are useful tools to study cell toxicity and perform high throughput screening of drugs. However, these models have several limitations including the cancerous properties of immortalized cell lines and the lack of authentic dopaminergic characteristics.^187,189 And due to the two-dimensional and single-cell culturing method, important cell-cell-interaction is lost. Therapeutic intervention or prevention of disease progression to this day still resembles the main challenge in PD and focusing on one subtype of cells is likely insufficient to predict clinical efficacy.¹⁹⁰

Organotypic brain slices

Tissue from genetically modified animals or PD patients can be cultured in an in vitro system called organotypic brain slices. This method preserves the physiological cell-to-cell interaction, by maintaining the architecture. Moreover, the simultaneously culturing of functionally related brain areas offers a unique tool to study aSyn spreading and seeding abilities.^191,192 For example, when injecting PFFs into the dentate gyrus of organotypic hippocampal brain slices, the aggregation of aSyn and its spreading to CA3 and CA1 regions can be observed.^193,194 To study dopaminergic circuitries, organotypic brain slices of the substantia nigra and striatum are established.¹⁹⁵ Moreover, the important interplay of neurons and glial cells during PD progression can be observed as well as neuronal activity measured via multielectrode array compatible culture methods.^196,197 Working with neurotoxins like MPTP and 6-OHDA, frequently used in animal models and in cellular models, has the disadvantage that aSyn pathology is missing and dopaminergic neurodegeneration is achieved only via oxidative stress induced by the toxin.¹⁹⁸ Mechanical transection of nigrostriatal pathways which initiates dopaminergic neurodegeneration is another method to induce neuronal loss in organotypic brain slices.¹⁹⁹ However, also this method lacks aSyn pathology.

Organotypic brain slices are often isolated from early postnatal mice or rats, but nowadays also protocols for adult mice/rat brain exist.^200,201 This allows preclinical testing in aged material with observable and measurable neuronal network activity over an extended period. Surgically resected human tissue and even postmortem tissue from humans can be used as donors.^202–204 This represents a powerful method to observe disease-associated pathology during culture.²⁰³ Overall organotypic brain slices obtained from animal models are useful additions to the in vivo studies and offer opportunities for human-tissue based preclinical drug development.²⁰³

IPSCs

Patient-derived inducible pluripotent stem cells (iPSCs) can be generated from cells taken, for example, as a skin biopsy of PD patients. These cells can be reprogrammed to embryonic stem cells with the complete genomic background of a patient and then used as cellular model with patient specific cells.²⁰⁵ Several protocols exist to differentiate stem cells into dopaminergic neurons, making it a suitable method for PD research.^206,207 IPSCs represent a very powerful tool, as it holds the ability of high throughput screening individualized to a patient.²⁰⁵ This in vitro strategy is often used to investigate how PD-associated mutations affect cellular pathways, since PD patients with known mutations were mostly used as donors.^208,209 Recently, sporadic PD patients are used as donors to understand the vulnerability of their neurons to the disease seeking to find common altered pathways which can be targeted by a drug.²¹⁰ For example, iPSCs from PD patients show abnormal accumulation of aSyn and downregulation of antioxidative pathways, making them sensitive for oxidative stress.²¹⁰ Although sporadic PD patients do not show a mutation associated with increased PD risk, iPSCs generated from this subgroup of patients show epigenetic changes which could add to their increased susceptibility.²⁰⁹ Increased oxidized dopamine levels and oxidative stress are also found in iPSCs generated from patients with mutations in genes associated with PD risk like SNCA, LRRK2, PINK1/Park2, and VPS35.^208,211

Overall, iPSCs do not only offer individualized screening for suitable therapeutical options, but they may also be suitable treatment options in the future.²¹² Allele-specific genome-targeted knock-in and knock-out via the CRISPR-Cas9 system allows regulation of endogenous gene expression and might lead to disease-resistant iPSCs as implants to replace the lost dopaminergic neurons.^213–216 A pitfall of iPSCs cultures is that they are for the most part lacking non-neuronal cells, such as microglia, astroglia or endothelial cells. There is ongoing research to overcome this limitation by co-culturing or creating complex organoids. Another criticism raised specifically for studying neurodegenerating diseases is that these cells do not replicate the impact of aging, as they remain relatively young neurons. However, in combination with other models iPSCs will further develop into a key in vitro system to develop therapy especially if specific genetic subtypes carrying certain mutations or risk alleles are targeted.

Organoids

The absence of 3D structure in in vitro systems means that the typical cell-cell-interactions of the brain, and the formation of a physiological cytoarchitecture, cannot be maintained. This is aggravated by the fact that the central nervous system is composed of several cell types that are usually not present in 2D culture systems. 3D systems based on iPSCs are being frequently used as so-called organoids, that self-organize to mimic the 3D structure of the brain.²¹⁷ This system allows the highest neuronal differentiation ex vivo and resembles a neuronal network with different subtypes of cells present in the brain.^218–220 Midbrain-like organoids are described to contain neuromelanin-positive, dopaminergic neurons and to form a functional, mature neuronal network.²²¹ Using iPSCs from human PD patients allows the differentiation of brain-like structure that mimic the key hallmarks of PD, like decreased dopaminergic neurons.²²² PD-specific aSyn pathology can be recapitulated when cells are derived from patients with SNCA triplications. Elevated levels of aSyn and phosphorylation of the protein was detected, while the organoid expressed increase senescent markers and reduced dopaminergic neurons.^223–225 As organoids also recapitulate rhythmic electrical activity, they are suitable to quantify alterations of neuronal activity, not only neuronal cell loss.²²⁶

Thus far organoids are focused on specific cell types, but efforts are made to create multi-cell type models that contain neurons, glia and aspects of the blood brain barrier. For example, organoids can be co-cultured with microglia precursor cells which also induces the development of vascular structures.²²⁷ Microfluidic chambers allow active perfusion of the necrotic organoid core and enable observation of cell-vessel interaction.^228,229 However, comparison of cortical cells and cortical organoids via high-throughput single-cell RNA sequencing revealed reduced subtype resolution due to lack of maturation.²³⁰ Similar to iPSCs, organoids model the embryonic development of the brain and lack age-dependent characteristics as well as myelination and microglia and oligodendroglial migration.^231–233 Not only aging but also maturation of synapses, dendrites and axonal projections are missing, making studies on nigrostriatal circuitries impossible.^234,235 Extending the culture period up to 250 days allows maturation of the organoid, but immense costs and low reproducibility restrict the utilization of this method.²³² Pharmacologically induction of aging in organoids alters cellular pathways which could be misleading when studying age dependent diseases like PD.^236,237

Uses and limitations of in vitro strategies

In vitro strategies are rapidly developing field in neuroscience and their continuous improvements will increase their usability in PD research. Key examples for uses and limitations are summarized in Table 2. Out of all limitations, the poor translational value remains one of the biggest obstacles of in vitro studies at the moment. Although in vitro models are instrumental for mechanistic/molecular studies, it is important to consider that, for example, immortalized cell lines carry cancerous characteristics that must be considered.¹⁸⁹ Even iPSCs, which from a genetic point of view, closely resemble the human they were generated from, lack the highly-relevant factor of age. Due to the reprogramming, cells are set back to the chronological age of almost foetal, which is observable in an increase length of telomerases.²³⁸ Effort is put into establishing iPSCs that express aging marker, but they still exhibit different mitochondrial arrangements and lower oxidative stress compared to adult cells.^239,240 Not only immortalized cell lines but also primary neurons and iPSCs show high variability among clones which makes comparison between studies and experiments hard. The described variabilities appear unrelated to PD-phenotype or genotype and is most likely due to differences in maintenance and isolation protocols.²⁴¹ Bridging knowledge gaps by combining in vitro and in vivo experiments might bring up promising therapeutical targets.²⁴² Beyond that, in vitro methods can be used strategically to reduce costs and ease ethical concerns when screening for drugs.

Table 2.

Uses and limitations of in vitro strategies.

In vitro methods	Uses	Limitations
Immortalized cell lines	− High throughput screening because of high proliferation rate − Cell viability assays using MPTP, 6-OHDA, and PFFs − aSyn aggregation assays	− Cancerous properties¹⁸⁹ − Not authentic dopaminergic neurons¹⁸⁷ − Lack of standardization²⁴¹
Primary neurons	− Transfering genetic modification of animal model into culture¹⁹⁰ − High translatability → Drug screening − Cell viability assays − Eletrically active → neuronal network analysis	− Not human origin − Not solely dopaminergic neurons
Organotypic brain slices	− aSyn seeding and spreading assays¹⁹³ − Neuronal network activity measurable¹⁹⁷ − Nerve fiber outgrow¹⁹⁹	− Too cost-intensive for high throughput screening¹⁹⁴ − Mostly isolated from early postnatal mice/rats and do not represent maturing and aging¹⁹⁴
iPSCs	− Patient-individualized screening for therapeutical targets²⁰⁵ − Patient-individualized studying of disease mechanisms	− Lack of standardization − Age as missing factor²³⁸ − Too cost-intensive for high throughput screening
Organoids	− 3D structure, self-organization into brain specific cells²¹⁷ − Highest neuronal differentiation ex vivo²²⁰	− Too cost-intensive for high throughput screening²³² − Lack of maturation²³⁴ − Lack of vascularization and other cell types^229,230

As modelling of PD is based on genetic or toxic manipulation in cell culture systems and in animal models, the in vitro methods suffer from similar limitations as the accompanying in vivo experiments. This includes the possibility to measure downstream alterations of the modification not associated with PD pathology or to induce compensatory mechanisms in the cells, which do not resemble the disease. When working with immortalized cell lines, the immortalization changes the cell genotype and translation to the human situation might not be reliable.¹⁹⁰

Lastly, one major drawback of in vitro and ex vivo strategies is the lack of vascularization, for example in organoid models. Crosstalk happening at the blood-brain-barrier and immune invasion are described to play a key role in PD progression.^187,243 When blood flow is missing, detecting possible biomarkers for blood analysis can only be achieved by measuring substances, secreted into the cell culture media.²⁴⁴ While organotypic brain slices show a network of laminin-positive brain capillaries, they are not functional in culture.²⁴⁵ Nonetheless, organotypic brain slice culture represents the closest to in vivo to study secretion of vascular molecules and response to angiogenic substances in culture.^246,247

Minding the limitations and uses, in vitro models should be included in experiments in PD research to improve our understanding of the disease and improve replacement methods. Overall, PD might be a cellular disease, indicated by functional loss of several cellular pathways including autophagic dysfunction, mitochondrial dysfunction, ER alterations.²⁴⁸ To reduce cost issues and low translation, more research into innovative strategies is necessary.¹⁸⁷

Conclusion: Do we still need animal models in PD research?

One of the major drawbacks of in vitro models is that they lack the ability to reproduce the complex architecture of the brain, its functional readout, and its connection to other organs. And while it is well-described and recognized that each in vivo model also lacks major parts of disease characteristics, they do not have these limitations. However, the advantages of emerging in vitro methods discussed above show the potential of reducing animal experiments in PD while simultaneously opening new possibilities to screen for therapeutics. Knowledge gaps in PD research might not be filled by sticking solely to animal models, but without them we will not be able to develop safe and efficacious drugs. Currently, aSyn pathology is the most investigated and tested drug target for disease-modification in PD, and while specific mechanisms can be further studied and validated in in vitro models, the complexity of its spreading and down-stream pathology requires a complete brain with all cell types connected in a functional neuronal network for finite conclusions and drug efficacy testing. New methods that simplify the complex disease at the level of single cells, might offer opportunities to detect cellular mechanisms not yet discovered.¹⁹⁰ Nonetheless, animal experiments will be needed in PD research to detect behavioural deficits replicating main clinical endpoints, which cannot be mimicked by in vitro methods.¹⁸⁸ Ultimately, we argue that multimodal approaches, combining the findings of different in vitro and in vivo models, will more likely lead to the identification and validation of the long-sought disease modifying strategies for treating PD and related disorders.

Footnotes

Acknowledgements

The authors are grateful to the many colleagues which, in past, present and future, work tirelessly to improve treatment of Parkinson's disease, using in vitro and in vivo models, thereby certainly adhering to 3R principles, which is part of good laboratory practice and ethical guidelines in research.

ORCID iDs

Kristina Lau

Tiago F Outeiro

Franziska Richter

Ethical considerations

We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. No approval was required from the local ethical committee for this review article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by intramural funding from the University of Veterinary Medicine Hannover.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Datasets/data availability statement

The raw data of this study are available on reasonable request from the corresponding author.

References

Ben-Shlomo

Darweesh

Llibre-Guerra

, et al. The epidemiology of Parkinson’s disease. Lancet 2024; 403: 283–292.

World Health Organization [WHO]. Parkinson disease, https://www.who.int/news-room/fact-sheets/detail/parkinson-disease (2023).

Berg

Postuma

Bloem

, et al. Time to redefine PD? Introductory statement of the MDS task force on the definition of Parkinson’s disease. Mov Disord 2014; 29: 454–462.

Postuma

Berg

Stern

, et al. MDS Clinical diagnostic criteria for Parkinson’s disease. Mov Disord 2015; 30: 1591–1601.

Hirsch

Graybiel

Duyckaerts

, et al. Neuronal loss in the pedunculopontine tegmental nucleus in Parkinson disease and in progressive supranuclear palsy. Proc Natl Acad Sci U S A 1987; 84: 5976–5980.

Gaspar

Duyckaerts

Alvarez

, et al. Alterations of dopaminergic and noradrenergic innervations in motor cortex in Parkinson’s disease. Ann Neurol 1991; 30: 365–374.

Halliday

Blumbergs

Cotton

, et al. Loss of brainstem serotonin- and substance P-containing neurons in Parkinson’s disease. Brain Res 1990; 510: 104–107.

Lange

Kornhuber

Riederer

. Dopamine/glutamate interactions in Parkinson’s disease. Neurosci Biobehav Rev 1997; 21: 393–400.

Postuma

Aarsland

Barone

, et al. Identifying prodromal Parkinson’s disease: pre-motor disorders in Parkinson’s disease. Mov Disord 2012; 27: 617–626.

10.

Yang

Hamilton

Kopil

, et al. Current and projected future economic burden of Parkinson’s disease in the U.S. NPJ Parkinsons Dis 2020; 6: 15.

11.

Bernstein

Stout

Miller

. The vesicular monoamine transporter 2: an underexplored pharmacological target. Neurochem Int 2014; 73: 89–97.

12.

Bispo

JMM

Melo

JEC

Gois

, et al. Sex differences in the progressive model of parkinsonism induced by reserpine in rats. Behav Brain Res 2019; 363: 23–29.

13.

Leao

Meurer

da Silva

, et al. Spontaneously hypertensive rats (SHR) are resistant to a reserpine-induced progressive model of Parkinson’s disease: differences in motor behavior, tyrosine hydroxylase and alpha-synuclein expression. Front Aging Neurosci 2017; 9: 78.

14.

Santos

Cunha

Dierschnabel

, et al. Cognitive, motor and tyrosine hydroxylase temporal impairment in a model of parkinsonism induced by reserpine. Behav Brain Res 2013; 253: 68–77.

15.

Carlsson

Lindqvist

Magnusson

. 3,4-Dihydroxyphenylalanine And 5-hydroxytryptophan as reserpine antagonists. Nature 1957; 180: 1200.

16.

Leal

Lins

de Gois

, et al. Commentary: evaluation of models of Parkinson’s disease. Front Neurosci 2016; 10: 283.

17.

Leao

Sarmento-Silva

Santos

, et al. Molecular, neurochemical, and behavioral hallmarks of reserpine as a model for Parkinson’s disease: new perspectives to a long-standing model. Brain Pathol 2015; 25: 377–390.

18.

Lama

Buhidma

Fletcher

EJR

, et al. Animal models of Parkinson’s disease: a guide to selecting the optimal model for your research. Neuronal Signal 2021; 5: NS20210026.

19.

Gardner

Baldessarini

Waraich

. Modern antipsychotic drugs: a critical overview. CMAJ 2005; 172: 1703–1711.

20.

Sanberg

. Haloperidol-induced catalepsy is mediated by postsynaptic dopamine receptors. Nature 1980; 284: 472–473.

21.

Kulkarni

Bishnoi

Chopra

. In vivo microdialysis studies of striatal level of neurotransmitters after haloperidol and chlorpromazine administration. Indian J Exp Biol 2009; 47: 91–97.

22.

Balijepalli

Kenchappa

Boyd

, et al. Protein thiol oxidation by haloperidol results in inhibition of mitochondrial complex I in brain regions: comparison with atypical antipsychotics. Neurochem Int 2001; 38: 425–435.

23.

Thakur

Prakash

Bisht

, et al. Beneficial effect of candesartan and lisinopril against haloperidol-induced tardive dyskinesia in rat. J Renin Angiotensin Aldosterone Syst 2015; 16: 917–929.

24.

Dovonou

Bolduc

Soto Linan

, et al. Animal models of Parkinson’s disease: bridging the gap between disease hallmarks and research questions. Transl Neurodegener 2023; 12: 36.

25.

Schober

. Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res 2004; 318: 215–224.

26.

Blum

Torch

Lambeng

, et al. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog Neurobiol 2001; 65: 135–172.

27.

Kirik

Rosenblad

Bjorklund

. Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp Neurol 1998; 152: 259–277.

28.

Stott

SRW

Barker

. Time course of dopamine neuron loss and glial response in the 6-OHDA striatal mouse model of Parkinson’s disease. Eur J Neurosci 2014; 39: 1042–1056.

29.

Branchi

D'Andrea

Armida

, et al. Nonmotor symptoms in Parkinson’s disease: investigating early-phase onset of behavioral dysfunction in the 6-hydroxydopamine-lesioned rat model. J Neurosci Res 2008; 86: 2050–2061.

30.

Colucci

Cervio

Faniglione

, et al. Intestinal dysmotility and enteric neurochemical changes in a Parkinson’s disease rat model. Auton Neurosci 2012; 169: 77–86.

31.

Pellegrini

Ippolito

Segnani

, et al. Pathological remodelling of colonic wall following dopaminergic nigrostriatal neurodegeneration. Neurobiol Dis 2020; 139: 104821.

32.

Nicklas

Youngster

Kindt

, et al. MPTP, MPP+ and mitochondrial function. Life Sci 1987; 40: 721–729.

33.

Ding

Luan

, et al. Development of a stable, early stage unilateral model of Parkinson’s disease in middle-aged rhesus monkeys. Exp Neurol 2008; 212: 431–439.

34.

Lofrumento

Saponaro

Cianciulli

, et al. MPTP-induced neuroinflammation increases the expression of pro-inflammatory cytokines and their receptors in mouse brain. Neuroimmunomodulation 2011; 18: 79–88.

35.

Liu

, et al. Phosphorylation of tau and alpha-synuclein induced neurodegeneration in MPTP mouse model of Parkinson’s disease. Neuropsychiatr Dis Treat 2020; 16: 651–663.

36.

Przedborski

Jackson-Lewis

Naini

, et al. The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a technical review of its utility and safety. J Neurochem 2001; 76: 1265–1274.

37.

Schmidt

Ferger

. Neurochemical findings in the MPTP model of Parkinson’s disease. J Neural Transm (Vienna) 2001; 108: 1263–1282.

38.

Mader

Pivtoraiko

Flippo

, et al. Rotenone inhibits autophagic flux prior to inducing cell death. ACS Chem Neurosci 2012; 3: 1063–1072.

39.

Betarbet

Sherer

MacKenzie

, et al. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 2000; 3: 1301–1306.

40.

Yuan

Yan

Sun

, et al. The molecular mechanism of rotenone-induced alpha-synuclein aggregation: emphasizing the role of the calcium/GSK3beta pathway. Toxicol Lett 2015; 233: 163–171.

41.

Inden

Kitamura

Takeuchi

, et al. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. J Neurochem 2007; 101: 1491–1504.

42.

Xiong

Jia

, et al. The role of autophagy in Parkinson’s disease: rotenone-based modeling. Behav Brain Funct 2013; 9: 13.

43.

Fleming

Zhu

Fernagut

, et al. Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone. Exp Neurol 2004; 187: 418–429.

44.

Greenamyre

Cannon

Drolet

, et al. Lessons from the rotenone model of Parkinson’s disease. Trends Pharmacol Sci 2010; 31: 141–142; author reply 142–143.

45.

Rappold

Cui

Chesser

, et al. Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3. Proc Natl Acad Sci U S A 2011; 108: 20766–20771.

46.

Cicchetti

Lapointe

Roberge-Tremblay

, et al. Systemic exposure to paraquat and maneb models early Parkinson’s disease in young adult rats. Neurobiol Dis 2005; 20: 360–371.

47.

McCormack

Thiruchelvam

Manning-Bog

, et al. Environmental risk factors and Parkinson’s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol Dis 2002; 10: 119–127.

48.

Manning-Bog

McCormack

, et al. The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein. J Biol Chem 2002; 277: 1641–1644.

49.

Hemmati

Hicks

. Increased myofibroblast contractile sensitivity in paraquat pretreated rat lung tissue. Life Sci 1999; 65: 2325–2332.

50.

Liu

Bing

. Lipopolysaccharide animal models for Parkinson’s disease. Parkinsons Dis 2011; 2011: 327089.

51.

Hunter

Dragicevic

Seifert

, et al. Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system. J Neurochem 2007; 100: 1375–1386.

52.

Hunter

Ojha

Bhurtel

, et al. Lipopolysaccharide-induced functional and structural injury of the mitochondria in the nigrostriatal pathway. Neurosci Res 2017; 114: 62–69.

53.

Choi

Zhang

Bing

. Aging enhances the neuroinflammatory response and alpha-synuclein nitration in rats. Neurobiol Aging 2010; 31: 1649–1653.

54.

Choi

Liu

Hunter

, et al. Striatal neuroinflammation promotes parkinsonism in rats. PLoS One 2009; 4: e5482.

55.

Zhang

Stanton

Nguyen

, et al. Intrapallidal lipopolysaccharide injection increases iron and ferritin levels in glia of the rat substantia nigra and induces locomotor deficits. Neuroscience 2005; 135: 829–838.

56.

Cooper

Machiela

Dues

, et al. Activation of the mitochondrial unfolded protein response promotes longevity and dopamine neuron survival in Parkinson’s disease models. Sci Rep 2017; 7: 16441.

57.

Greene

Whitworth

Kuo

, et al. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc Natl Acad Sci U S A 2003; 100: 4078–4083.

58.

Dawson

. PINK1 And parkin mitochondrial quality control: a source of regional vulnerability in Parkinson’s disease. Mol Neurodegener 2020; 15: 20.

59.

Yang

. A CRISPR monkey model unravels a unique function of PINK1 in primate brains. Mol Neurodegener 2019; 14: 17.

60.

Wallings

Manzoni

Bandopadhyay

. Cellular processes associated with LRRK2 function and dysfunction. FEBS J 2015; 282: 2806–2826.

61.

Beccano-Kelly

Volta

Munsie

, et al. LRRK2 Overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Hum Mol Genet 2015; 24: 1336–1349.

62.

Yue

Hinkle

Davies

, et al. Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice. Neurobiol Dis 2015; 78: 172–195.

63.

Bardai

Ordonez

Bailey

, et al. Lrrk promotes tau neurotoxicity through dysregulation of actin and mitochondrial dynamics. PLoS Biol 2018; 16: e2006265.

64.

Melrose

Dachsel

Behrouz

, et al. Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice. Neurobiol Dis 2010; 40: 503–517.

65.

Liu

, et al. Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson’s disease. Nat Neurosci 2009; 12: 826–828.

66.

Aleyasin

Rousseaux

Marcogliese

, et al. DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway. Proc Natl Acad Sci U S A 2010; 107: 3186–3191.

67.

Chen

Cagniard

Mathews

, et al. Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice. J Biol Chem 2005; 280: 21418–21426.

68.

Kang

Chen

, et al. DJ-1 Inhibits alpha-synuclein aggregation by regulating chaperone-mediated autophagy. Front Aging Neurosci 2017; 9: 308.

69.

Lowe

McDermott

Landon

, et al. Ubiquitin carboxyl-terminal hydrolase (PGP 9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. J Pathol 1990; 161: 153–160.

70.

Miura

Oda

Endo

, et al. Progressive degeneration of motor nerve terminals in GAD mutant mouse with hereditary sensory axonopathy. Neuropathol Appl Neurobiol 1993; 19: 41–51.

71.

Oda

Yamazaki

Miura

, et al. Dying back type axonal degeneration of sensory nerve terminals in muscle spindles of the gracile axonal dystrophy (GAD) mutant mouse. Neuropathol Appl Neurobiol 1992; 18: 265–281.

72.

Cerqueira

von Stockum

Giacomello

, et al. A new target for an old DUB: UCH-L1 regulates mitofusin-2 levels, altering mitochondrial morphology, function and calcium uptake. Redox Biol 2020; 37: 101676.

73.

Setsuie

Wang

Mochizuki

, et al. Dopaminergic neuronal loss in transgenic mice expressing the Parkinson’s disease-associated UCH-L1 I93M mutant. Neurochem Int 2007; 50: 119–129.

74.

Taguchi

Liu

Ruan

, et al. Glucosylsphingosine promotes alpha-synuclein pathology in mutant GBA-associated Parkinson’s disease. J Neurosci 2017; 37: 9617–9631.

75.

Ginns

Mak

, et al. Neuroinflammation and alpha-synuclein accumulation in response to glucocerebrosidase deficiency are accompanied by synaptic dysfunction. Mol Genet Metab 2014; 111: 152–162.

76.

Ham

, et al. Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations. Autophagy 2019; 15: 113–130.

77.

Abul Khair

Dhanushkodi

Ardah

, et al. Silencing of glucocerebrosidase gene in Drosophila enhances the aggregation of Parkinson’s disease associated alpha-synuclein mutant A53T and affects locomotor activity. Front Neurosci 2018; 12: 81.

78.

Farfel-Becker

Tayebi

, et al.

Can GBA1-associated Parkinson disease be modeled in the mouse?

Trends Neurosci 2019; 42: 631–643.

79.

Rockenstein

Mallory

Hashimoto

, et al. Differential neuropathological alterations in transgenic mice expressing alpha-synuclein from the platelet-derived growth factor and thy-1 promoters. J Neurosci Res 2002; 68: 568–578.

80.

Subramaniam

Vergnes

Franich

, et al. Region specific mitochondrial impairment in mice with widespread overexpression of alpha-synuclein. Neurobiol Dis 2014; 70: 204–213.

81.

Watson

Richter

Lee

, et al. Regionally-specific microglial activation in young mice over-expressing human wildtype alpha-synuclein. Exp Neurol 2012; 237: 318–334.

82.

Chesselet

Richter

. Modelling of Parkinson’s disease in mice. Lancet Neurol 2011; 10: 1108–1118.

83.

Chesselet

Richter

Zhu

, et al. A progressive mouse model of Parkinson’s disease: the Thy1-aSyn (“line 61”) mice. Neurotherapeutics 2012; 9: 297–314.

84.

Richter

Stanojlovic

Kaufer

, et al. A mouse model to test novel therapeutics for Parkinson’s disease: an update on the Thy1-aSyn (“line 61”) mice. Neurotherapeutics 2023; 20: 97–116.

85.

Wegrzynowicz

Bar-On

Calo

, et al. Depopulation of dense alpha-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson’s disease model. Acta Neuropathol 2019; 138: 575–595.

86.

Zarranz

Alegre

Gomez-Esteban

, et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 2004; 55: 164–173.

87.

Emmer

Waxman

Covy

, et al. E46k human alpha-synuclein transgenic mice develop Lewy-like and tau pathology associated with age-dependent, detrimental motor impairment. J Biol Chem 2011; 286: 35104–35118.

88.

Piltonen

Savolainen

Patrikainen

, et al. Comparison of motor performance, brain biochemistry and histology of two A30P alpha-synuclein transgenic mouse strains. Neuroscience 2013; 231: 157–168.

89.

Poon

Frasier

Shreve

, et al. Mitochondrial associated metabolic proteins are selectively oxidized in A30P alpha-synuclein transgenic mice–a model of familial Parkinson’s disease. Neurobiol Dis 2005; 18: 492–498.

90.

Giasson

Duda

Quinn

, et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron 2002; 34: 521–533.

91.

Chung

Perez-Acuna

, et al. Modeling alpha-synuclein propagation with preformed fibril injections. J Mov Disord 2019; 12: 139–151.

92.

Luk

Kehm

Carroll

, et al. Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 2012; 338: 949–953.

93.

Sorrentino

Xia

Funk

, et al. Motor neuron loss and neuroinflammation in a model of alpha-synuclein-induced neurodegeneration. Neurobiol Dis 2018; 120: 98–106.

94.

Sun

, et al. Comparison between alpha-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model. Biochem Biophys Res Commun 2015; 464: 988–993.

95.

Lo Bianco

Ridet

Schneider

, et al. Alpha -synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc Natl Acad Sci U S A 2002; 99: 10813–10818.

96.

Kirik

Rosenblad

Burger

, et al. Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci 2002; 22: 2780–2791.

97.

Klaus

Karikari

, et al. AAV1/2-induced Overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson’s disease. Acta Neuropathol Commun 2017; 5: 11.

98.

Chung

Koprich

Siddiqi

, et al. Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy. J Neurosci 2009; 29: 3365–3373.

99.

Di Maio

Barrett

Hoffman

, et al. . Alpha-synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease. Sci Transl Med 2016; 8: 342–378.

100.

McKinnon

De Snoo

Gondard

, et al. Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by alpha-synuclein. Acta Neuropathol Commun 2020; 8: 17.

101.

Berard

Sheta

Malvaut

, et al. A light-inducible protein clustering system for in vivo analysis of alpha-synuclein aggregation in Parkinson disease. PLoS Biol 2022; 20: e3001578.

102.

Huntington

Srinivasan

. Adeno-Associated virus expression of alpha-synuclein as a tool to model Parkinson’s disease: current understanding and knowledge gaps. Aging Dis 2021; 12: 1120–1137.

103.

Zucca

Segura-Aguilar

Ferrari

, et al. Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson’s disease. Prog Neurobiol 2017; 155: 96–119.

104.

Viceconte

Burguillos

Herrera

, et al. Neuromelanin activates proinflammatory microglia through a caspase-8-dependent mechanism. J Neuroinflammation 2015; 12: 5.

105.

Carballo-Carbajal

Laguna

Romero-Gimenez

, et al. Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis. Nat Commun 2019; 10: 973.

106.

Collaborators GBDPsD. Global, regional, and national burden of Parkinson’s disease, 1990-2016: a systematic analysis for the global burden of disease study 2016. Lancet Neurol 2018; 17: 939–953.

107.

Carvey

Punati

Newman

. Progressive dopamine neuron loss in Parkinson’s disease: the multiple hit hypothesis. Cell Transplant 2006; 15: 239–250.

108.

Miller

O'Callaghan

. Do early-life insults contribute to the late-life development of Parkinson and Alzheimer diseases? Metabolism 2008; 57: S44–S49.

109.

Polymeropoulos

Lavedan

Leroy

, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 1997; 276: 2045–2047.

110.

Healy

Falchi

O'Sullivan

, et al. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson’s disease: a case-control study. Lancet Neurol 2008; 7: 583–590.

111.

Ling

Gayle

, et al. In utero bacterial endotoxin exposure causes loss of tyrosine hydroxylase neurons in the postnatal rat midbrain. Mov Disord 2002; 17: 116–124.

112.

Martyn

Osmond

. Parkinson’s disease and the environment in early life. J Neurol Sci 1995; 132: 201–206.

113.

Langston

Ballard

Tetrud

, et al. Chronic parkinsonism in humans due to a product of meperidine-analog synthesis. Science 1983; 219: 979–980.

114.

Elbaz

Clavel

Rathouz

, et al. Professional exposure to pesticides and Parkinson disease. Ann Neurol 2009; 66: 494–504.

115.

Davis

Williams

Markey

, et al. Chronic parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res 1979; 1: 249–254.

116.

Schapira

Cooper

Dexter

, et al. Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 1990; 54: 823–827.

117.

Nicklas

Vyas

Heikkila

. Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. Life Sci 1985; 36: 2503–2508.

118.

Markey

Johannessen

Chiueh

, et al. Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism. Nature 1984; 311: 464–467.

119.

Giovanni

Sieber

Heikkila

, et al. Studies on species sensitivity to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Part 1: systemic administration. J Pharmacol Exp Ther 1994; 270: 1000–1007.

120.

Richter

Gabby

McDowell

, et al. Effects of decreased dopamine transporter levels on nigrostriatal neurons and paraquat/maneb toxicity in mice. Neurobiol Aging 2017; 51: 54–66.

121.

McCormack

Atienza

Johnston

, et al. Role of oxidative stress in paraquat-induced dopaminergic cell degeneration. J Neurochem 2005; 93: 1030–1037.

122.

Costello

Cockburn

Bronstein

, et al. Parkinson’s disease and residential exposure to maneb and paraquat from agricultural applications in the central valley of California. Am J Epidemiol 2009; 169: 919–926.

123.

Cannon

Greenamyre

. Neurotoxic in vivo models of Parkinson’s disease recent advances. Prog Brain Res 2010; 184: 17–33.

124.

Mercado

Käufer

Richter

, et al. Infections in the etiology of Parkinson’s disease and synucleinopathies: a renewed perspective, mechanistic insights and therapeutic implications. J Parkinsons Dis 2024; 14: 1301–1329.

125.

Marreiros

Muller-Schiffmann

Trossbach

, et al. Disruption of cellular proteostasis by H1N1 influenza A virus causes alpha-synuclein aggregation. Proc Natl Acad Sci U S A 2020; 117: 6741–6751.

126.

Philippens

Boszormenyi

Wubben

JAM

, et al. Brain Inflammation and Intracellular alpha-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection. Viruses 2022; 14: 776.

127.

Käufer

Schreiber

Hartke

A-S

, et al. Microgliosis and neuronal proteinopathy in brain persist beyond viral clearance in SARS-CoV-2 hamster model. eBioMedicine 2022; 79: 103999.

128.

Schreiber

Wiesweg

Stanelle-Bertram

, et al. Sex-specific biphasic alpha-synuclein response and alterations of interneurons in a COVID-19 hamster model. EBioMedicine 2024; 105: 105191.

129.

Thi Lai

Kim

Nguyen

LTN

, et al. Microglial inhibition alleviates alpha-synuclein propagation and neurodegeneration in Parkinson’s disease mouse model. NPJ Parkinsons Dis 2024; 10: 32.

130.

Hartke

Schreiber

Lau

, et al. Alpha-synuclein pathology enhances peripheral and CNS immune responses to bacterial endotoxins. Neurobiol Dis 2025; 205: 106773.

131.

Brown

Camacho

Williams-Gray

. The endotoxin hypothesis of Parkinson’s disease. Mov Disord 2023; 38: 1143–1155.

132.

Deng

Wiese

Zhou

, et al. The efficacy of systemic administration of lipopolysaccharide in modelling pre-motor Parkinson’s disease in C57BL/6 mice. Neurotoxicology 2021; 85: 254–264.

133.

Gorecki

Preskey

Bakeberg

, et al. Altered gut microbiome in Parkinson’s disease and the influence of lipopolysaccharide in a human alpha-synuclein over-expressing mouse model. Front Neurosci 2019; 13: 839.

134.

Paolicelli

Sierra

Stevens

, et al. Microglia states and nomenclature: a field at its crossroads. Neuron 2022; 110: 3458–3483.

135.

Spillantini

Crowther

Jakes

, et al. Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci U S A 1998; 95: 6469–6473.

136.

Braak

Del Tredici

Bratzke

, et al. Staging of the intracerebral inclusion body pathology associated with idiopathic Parkinson’s disease (preclinical and clinical stages). J Neurol 2002; 249: III/1–III/5.

137.

Braak

Rub

Gai

, et al. Idiopathic Parkinson’s disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm (Vienna) 2003; 110: 517–536.

138.

Jakes

Spillantini

Goedert

. Identification of two distinct synucleins from human brain. FEBS Lett 1994; 345: 27–32.

139.

Fauvet

Mbefo

Fares

, et al. . Alpha-synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer. J Biol Chem 2012; 287: 15345–15364.

140.

Weinreb

Zhen

Poon

, et al. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 1996; 35: 13709–13715.

141.

Serpell

Berriman

Jakes

, et al. Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc Natl Acad Sci U S A 2000; 97: 4897–4902.

142.

Desplats

Lee

Bae

, et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci U S A 2009; 106: 13010–13015.

143.

Fujiwara

Hasegawa

Dohmae

, et al. Alpha-synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol 2002; 4: 160–164.

144.

Kuzuhara

Mori

Izumiyama

, et al. Lewy bodies are ubiquitinated. A light and electron microscopic immunocytochemical study. Acta Neuropathol 1988; 75: 345–353.

145.

Baba

Nakajo

, et al. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 1998; 152: 879–884.

146.

Vicente Miranda

Szego

Oliveira

LMA

, et al. Glycation potentiates alpha-synuclein-associated neurodegeneration in synucleinopathies. Brain 2017; 140: 1399–1419.

147.

van der Putten

Wiederhold

Probst

, et al. Neuropathology in mice expressing human alpha-synuclein. J Neurosci 2000; 20: 6021–6029.

148.

Betarbet

Sherer

Greenamyre

. Animal models of Parkinson’s disease. Bioessays 2002; 24: 308–318.

149.

Cabin

Gispert-Sanchez

Murphy

, et al. Exacerbated synucleinopathy in mice expressing A53 T SNCA on a snca null background. Neurobiol Aging 2005; 26: 25–35.

150.

Volpicelli-Daley

Luk

Patel

, et al. Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 2011; 72: 57–71.

151.

Challis

Hori

Sampson

, et al. Gut-seeded alpha-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice. Nat Neurosci 2020; 23: 327–336.

152.

Recasens

Dehay

Bove

, et al. Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys. Ann Neurol 2014; 75: 351–362.

153.

Peelaerts

Bousset

Van der Perren

, et al. alpha-Synuclein strains cause distinct synucleinopathies after local and systemic administration. Nature 2015; 522: 340–344.

154.

Winner

Jappelli

Maji

, et al. In vivo demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A 2011; 108: 4194–4199.

155.

McNaught

Belizaire

Isacson

, et al. Altered proteasomal function in sporadic Parkinson’s disease. Exp Neurol 2003; 179: 38–46.

156.

Valente

Salvi

Ialongo

, et al. PINK1 Mutations are associated with sporadic early-onset parkinsonism. Ann Neurol 2004; 56: 336–341.

157.

Tanji

Mori

Kakita

, et al. Alteration of autophagosomal proteins (LC3, GABARAP and GATE-16) in Lewy body disease. Neurobiol Dis 2011; 43: 690–697.

158.

Dehay

Bove

Rodriguez-Muela

, et al. Pathogenic lysosomal depletion in Parkinson’s disease. J Neurosci 2010; 30: 12535–12544.

159.

Goker-Alpan

Schiffmann

LaMarca

, et al. Parkinsonism among gaucher disease carriers. J Med Genet 2004; 41: 937–940.

160.

Sidransky

Nalls

Aasly

, et al. Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N Engl J Med 2009; 361: 1651–1661.

161.

Mazzulli

Sun

, et al. Gaucher disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell 2011; 146: 37–52.

162.

Bourdenx

Bezard

Dehay

. Lysosomes and alpha-synuclein form a dangerous duet leading to neuronal cell death. Front Neuroanat 2014; 8: 83.

163.

Sardi

Viel

Clarke

, et al. Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models. Proc Natl Acad Sci U S A 2017; 114: 2699–2704.

164.

Sardi

Singh

Cheng

, et al. Mutant GBA1 expression and synucleinopathy risk: first insights from cellular and mouse models. Neurodegener Dis 2012; 10: 195–202.

165.

Sardi

Clarke

Kinnecom

, et al. CNS Expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of gaucher-related synucleinopathy. Proc Natl Acad Sci U S A 2011; 108: 12101–12106.

166.

Schidlitzki

Stanojlovic

Fournier

, et al. Double-Edged effects of venglustat on behavior and pathology in mice overexpressing alpha-synuclein. Mov Disord 2023; 38: 1044–1055.

167.

Migdalska-Richards

Daly

Bezard

, et al. Ambroxol effects in glucocerebrosidase and alpha-synuclein transgenic mice. Ann Neurol 2016; 80: 766–775.

168.

Hornykiewicz

. [The tropical localization and content of noradrenalin and dopamine (3-hydroxytyramine) in the substantia nigra of normal persons and patients with Parkinson’s disease. Wien Klin Wochenschr 1963; 75: 309–312.

169.

Sachs

Jonsson

. Mechanisms of action of 6-hydroxydopamine. Biochem Pharmacol 1975; 24: 1–8.

170.

Ungerstedt

. 6-Hydroxy-dopamine Induced degeneration of central monoamine neurons. Eur J Pharmacol 1968; 5: 107–110.

171.

Perese

Ulman

Viola

, et al. A 6-hydroxydopamine-induced selective parkinsonian rat model. Brain Res 1989; 494: 285–293.

172.

Schwarting

Huston

. The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol 1996; 50: 275–331.

173.

Heikkila

Cohen

. In vivo generation of hydrogen peroxide from 6-hydroxydopamine. Experientia 1972; 28: 1197–1198.

174.

Graham

. Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol 1978; 14: 633–643.

175.

Von Voigtlander

Moore

. Turning behavior of mice with unilateral 6-hydroxydopamine lesions in the striatum: effects of apomorphine, L-DOPA, amanthadine, amphetamine and other psychomotor stimulants. Neuropharmacology 1973; 12: 451–462.

176.

Ungerstedt

Arbuthnott

. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res 1970; 24: 485–493.

177.

Lundblad

Usiello

Carta

, et al. Pharmacological validation of a mouse model of l-DOPA-induced dyskinesia. Exp Neurol 2005; 194: 66–75.

178.

Winkler

Kirik

Bjorklund

, et al. L-DOPA-induced dyskinesia in the intrastriatal 6-hydroxydopamine model of Parkinson’s disease: relation to motor and cellular parameters of nigrostriatal function. Neurobiol Dis 2002; 10: 165–186.

179.

Cenci

Lee

Bjorklund

. L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA. Eur J Neurosci 1998; 10: 2694–2706.

180.

Grosso Jasutkar

Mouradian

. Therapeutics in the pipeline targeting alpha-synuclein for Parkinson’s disease. Pharmacol Rev 2022; 74: 207–237.

181.

Vijiaratnam

Foltynie

. Disease modifying therapies III: novel targets. Neuropharmacology 2021; 201: 108839.

182.

Devos

Hirsch

Wyse

. Seven solutions for neuroprotection in Parkinson’s disease. Mov Disord 2021; 36: 306–316.

183.

Dawson

Golde

Lagier-Tourenne

. Animal models of neurodegenerative diseases. Nat Neurosci 2018; 21: 1370–1379.

184.

McDowell

Chesselet

. Animal models of the non-motor features of Parkinson’s disease. Neurobiol Dis 2012; 46: 597–606.

185.

McFarthing

Buff

Rafaloff

, et al. Parkinson’s disease drug therapies in the clinical trial pipeline: 2024 update. J Parkinsons Dis 2024; 14: 899–912.

186.

Chopra

Lang

Hoglinger

, et al. Towards a biological diagnosis of PD. Parkinsonism Relat Disord 2024; 122: 106078.

187.

Lal

Singh

Watts

, et al. Experimental models of Parkinson’s disease: challenges and opportunities. Eur J Pharmacol 2024; 980: 176819.

188.

Vitale

Manciocco

Alleva

. The 3R principle and the use of non-human primates in the study of neurodegenerative diseases: the case of Parkinson’s disease. Neurosci Biobehav Rev 2009; 33: 33–47.

189.

Krishna

Biryukov

Trefois

, et al. Systems genomics evaluation of the SH-SY5Y neuroblastoma cell line as a model for Parkinson’s disease. BMC genomics 2014; 15: 1154.

190.

Falkenburger

Saridaki

Dinter

. Cellular models for Parkinson’s disease. J Neurochem 2016; 139: 121–130.

191.

Humpel

. Organotypic brain slice cultures: a review. Neuroscience 2015; 305: 86–98.

192.

Woodhams

Atkinson

. Regeneration of entorhino-dentate projections in organotypic slice cultures: mode of axonal regrowth and effects of growth factors. Exp Neurol 1996; 140: 68–78.

193.

Elfarrash

Jensen

Ferreira

, et al. Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates. Acta Neuropathol Commun 2019; 7: 213.

194.

Elfarrash

Jensen

. Organotypic hippocampal slices, an emerging tool to model synucleinopathies. Neural Regen Res 2021; 16: 999–1000.

195.

Jaeger

Gonzalo Ruiz

Llinas

. Organotypic slice cultures of dopaminergic neurons of substantia nigra. Brain Res Bull 1989; 22: 981–991.

196.

Ucar

Stefanova

Humpel

. Spreading of aggregated alpha-synuclein in sagittal organotypic mouse brain slices. Biomolecules 2022; 12: 163.

197.

Berdichevsky

Sabolek

Levine

, et al. Microfluidics and multielectrode array-compatible organotypic slice culture method. J Neurosci Methods 2009; 178: 59–64.

198.

Cavaliere

Vicente

Matute

. An organotypic culture model to study nigro-striatal degeneration. J Neurosci Methods 2010; 188: 205–212.

199.

Daviaud

Garbayo

Lautram

, et al. Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson’s disease. Neuroscience 2014; 256: 10–22.

200.

McCaughey-Chapman

Connor

. Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system. J Neurosci Methods 2017; 277: 83–87.

201.

Mayerl

Ffrench-Constant

. Establishing an adult mouse brain hippocampal organotypic slice culture system that allows for tracing and pharmacological manipulation of ex vivo neurogenesis. Bio Protoc 2021; 11: e3869.

202.

Mewes

Franke

Singer

. Organotypic brain slice cultures of adult transgenic P301S mice–a model for tauopathy studies. PLoS One 2012; 7: e45017.

203.

Plug

Revers

Breur

, et al. Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies. Acta Neuropathol Commun 2024; 12: 83.

204.

Andersson

Avaliani

Svensson

, et al. Optogenetic control of human neurons in organotypic brain cultures. Sci Rep 2016; 6: 24818.

205.

Park

Arora

Huo

, et al. Disease-specific induced pluripotent stem cells. Cell 2008; 134: 877–886.

206.

Soldner

Hockemeyer

Beard

, et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 2009; 136: 964–977.

207.

Chambers

Fasano

Papapetrou

, et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 2009; 27: 275–280.

208.

Byers

Cord

Nguyen

, et al. SNCA Triplication Parkinson’s patient’s iPSC-derived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress. PLoS One 2011; 6: e26159.

209.

Fernandez-Santiago

Carballo-Carbajal

Castellano

, et al. Aberrant epigenome in iPSC-derived dopaminergic neurons from Parkinson’s disease patients. EMBO Mol Med 2015; 7: 1529–1546.

210.

Chang

Lee-Chen

, et al. Impairment of proteasome and anti-oxidative pathways in the induced pluripotent stem cell model for sporadic Parkinson’s disease. Parkinsonism Relat Disord 2016; 24: 81–88.

211.

Prots

Grosch

Brazdis

, et al. alpha-Synuclein oligomers induce early axonal dysfunction in human iPSC-based models of synucleinopathies. Proc Natl Acad Sci U S A 2018; 115: 7813–7818.

212.

Stoddard-Bennett

Reijo Pera

. Treatment of Parkinson’s disease through personalized medicine and induced pluripotent stem cells. Cells 2019; 8: 26.

213.

Smith

Abalde-Atristain

, et al. Efficient and allele-specific genome editing of disease loci in human iPSCs. Mol Ther 2015; 23: 570–577.

214.

Qing

Walter

Jarazo

, et al. CRISPR/Cas9 and piggyBac-mediated footprint-free LRRK2-G2019S knock-in reveals neuronal complexity phenotypes and alpha-synuclein modulation in dopaminergic neurons. Stem Cell Res 2017; 24: 44–50.

215.

Kantor

Tagliafierro

, et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol Ther 2018; 26: 2638–2649.

216.

Soldner

Stelzer

Shivalila

, et al. Parkinson-associated risk variant in distal enhancer of alpha-synuclein modulates target gene expression. Nature 2016; 533: 95–99.

217.

Kelava

Lancaster

. Dishing out mini-brains: current progress and future prospects in brain organoid research. Dev Biol 2016; 420: 199–209.

218.

Fatehullah

Tan

Barker

. Organoids as an in vitro model of human development and disease. Nat Cell Biol 2016; 18: 246–254.

219.

Haycock

. 3D Cell culture: a review of current approaches and techniques. Methods Mol Biol 2011; 695: 1–15.

220.

Kim

Choi

D'Avanzo

, et al. A 3D human neural cell culture system for modeling Alzheimer’s disease. Nat Protoc 2015; 10: 985–1006.

221.

Xiao

Sun

, et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 2016; 19: 248–257.

222.

Smits

Reinhardt

, et al. Modeling Parkinson’s disease in midbrain-like organoids. NPJ Parkinsons Dis 2019; 5: 5.

223.

Yang

Tran

, et al. Lewy body-like inclusions in human midbrain organoids carrying glucocerebrosidase and alpha-synuclein mutations. Ann Neurol 2021; 90: 490–505.

224.

Mohamed

Sirois

Ramamurthy

, et al. Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy. Brain Commun 2021; 3: fcab223.

225.

Muwanigwa

Modamio-Chamarro

Antony

PMA

, et al. Alpha-synuclein pathology is associated with astrocyte senescence in a midbrain organoid model of familial Parkinson’s disease. Mol Cell Neurosci 2024; 128: 103919.

226.

Tasnim

Liu

. Emerging bioelectronics for brain organoid electrophysiology. J Mol Biol 2022; 434: 167165.

227.

Worsdorfer

Dalda

Kern

, et al. Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells. Sci Rep 2019; 9: 15663.

228.

Salmon

Grebenyuk

Abdel Fattah

, et al. Engineering neurovascular organoids with 3D printed microfluidic chips. Lab Chip 2022; 22: 1615–1629.

229.

Urrestizala-Arenaza

Cerchio

Cavaliere

, et al. Limitations of human brain organoids to study neurodegenerative diseases: a manual to survive. Front Cell Neurosci 2024; 18: 1419526.

230.

Bhaduri

Andrews

Mancia Leon

, et al. Cell stress in cortical organoids impairs molecular subtype specification. Nature 2020; 578: 142–148.

231.

Andrews

Kriegstein

. Challenges of organoid research. Annu Rev Neurosci 2022; 45: 23–39.

232.

Quadrato

Brown

Arlotta

. The promises and challenges of human brain organoids as models of neuropsychiatric disease. Nat Med 2016; 22: 1220–1228.

233.

Tomassy

Dershowitz

Arlotta

. Diversity matters: a revised guide to myelination. Trends Cell Biol 2016; 26: 135–147.

234.

Sulzer

Surmeier

. Neuronal vulnerability, pathogenesis, and Parkinson’s disease. Mov Disord 2013; 28: 41–50.

235.

Zhong

Zhang

Fan

, et al. A single-cell RNA-Seq survey of the developmental landscape of the human prefrontal cortex. Nature 2018; 555: 524–528.

236.

Kim

Padmashri

, et al. Pluripotent stem cell-derived cerebral organoids reveal human oligodendrogenesis with dorsal and ventral origins. Stem Cell Rep 2019; 12: 890–905.

237.

Vera

Bosco

Studer

. Generating late-onset human iPSC-based disease models by inducing neuronal age-related phenotypes through telomerase manipulation. Cell Rep 2016; 17: 1184–1192.

238.

Suhr

Chang

Rodriguez

, et al. Telomere dynamics in human cells reprogrammed to pluripotency. PLoS One 2009; 4: e8124.

239.

Rohani

Johnson

Arnold

, et al.

The aging signature: a hallmark of induced pluripotent stem cells?

Aging Cell 2014; 13: 2–7.

240.

Miller

Ganat

Kishinevsky

, et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 2013; 13: 691–705.

241.

Xicoy

Wieringa

Martens

. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegener 2017; 12: 10.

242.

Slanzi

Iannoto

Rossi

, et al. In vitro models of neurodegenerative diseases. Front Cell Dev Biol 2020; 8: 328.

243.

Lau

Kotzur

Richter

. Blood-brain barrier alterations and their impact on Parkinson’s disease pathogenesis and therapy. Transl Neurodegener 2024; 13: 37.

244.

Simons

Fleming

. Role of rodent models in advancing precision medicine for Parkinson’s disease. Handb Clin Neurol / Edited by PJ Vinken and GW Bruyn 2023; 193: 3–16.

245.

Moser

Schmidt-Kastner

Hinterhuber

, et al. Brain capillaries and cholinergic neurons persist in organotypic brain slices in the absence of blood flow. Eur J Neurosci 2003; 18: 85–94.

246.

Moser

Reindl

Blasig

, et al. Brain capillary endothelial cells proliferate in response to NGF, express NGF receptors and secrete NGF after inflammation. Brain Res 2004; 1017: 53–60.

247.

Mishra

O'Farrell

Reynell

, et al. Imaging pericytes and capillary diameter in brain slices and isolated retinae. Nat Protoc 2014; 9: 323–336.

248.

Panicker

Dawson

, et al. The cell biology of Parkinson’s disease. J Cell Biol 2021; 220. DOI: 10.1083/jcb.202012095